PROJECT SUMMARY Current therapy cannot stop the growth of obliterating hyper-proliferative lesions in the lung vessels of severe pulmonary arterial hypertension (PAH) patients, because they are focused on improving heart function and pulmonary hemodynamics. This is a major clinical barrier impacting morbidity in severe PAH patients. Therefore, this proposal is focused on reducing or preventing abnormal vascular remodeling. To accomplish this objective, we propose to address the following questions: Is the inhibitor of differentiation protein 3 (ID3) a determinant of hyper-proliferative vascular lesions in PAH patients? And how ID3 epigenetically reprograms vascular stem cells to produce hyper-proliferative lesions? Such a focus on the role of ID3 in pulmonary vascular remodeling is based on our novel discoveries: ID3 contributes in the acquisition of vascular stem cells through endothelial-mesenchymal transition and abnormal remodeling of vessels. Higher ID3 expression was observed in arterioles of clinical human PAH lung samples. ID3 is bound to the transcription factor-ZRF1 in vascular stem cells. ID3-ZRF1 interactome controls the transcription of a growth driving long non-coding RNA-MALAT1. CRISPR/Cas knockout of MALAT1 expression inhibited the growth of vascular lesions. Based on these findings we hypothesize that ID3 mediated epigenetic reprogramming is a driver of hyper-proliferative vascular remodeling in PAH patients. We will test this hypothesis by the following inter-related specific aims. In Aim 1 we will confirm that ID3 via interaction with an epigenetic regulator ZRF1 reprograms pulmonary vascular stem cells. In Aim 2 we will investigate the functional role the ID3/ZRF1 complex in the transcriptional activation of lncRNA MALAT1 that drives hyper-proliferative growth. In Aim 3 we will determine the clinical relevance of ID3 in intimal lesions in the arterioles of clinical human lung PAH tissues. This study is innovative because it: (i) proposes to identify the novel mechanism showing ID3 epigenetically reprograms vascular stem cells to produce hyper- proliferative lesions; (ii) will apply cutting-edge mechanistic molecular approaches using PAH patient cells and pulmonary vascular stem cells, clinical PAH lung biopsy samples, and experimental models to achieve the objectives of this study. The proposed project is significant because (i) knowledge gained from this study is important for understanding the molecular mechanism of arterial hyper-proliferative remodeling in the lung of PAH patients. (ii) Furthermore the proposed research will allow underrepresented minority students at the FIU Stempel College of Public Health to engage in hands-on cutting edge novel molecular epigenetic research to ascertain new insight into the mechanism by which ID3 may contribute in the acquisition of abnormal pulmonary vascular stem cells, who would otherwise lack such opportunities. Furthermore, findings of this study will set the stage for testing of (i) ID3 as a molecular risk factor for severe PAH patients and (ii) developing ID3 antagonists for the prevention and treatment of PAH.